In Vivo Transdermal Multi-Ion Monitoring with a Potentiometric Microneedle-Based Sensor Patch

Microneedle sensor technology offers exciting opportunities for decentralized clinical analyses. A novel issue puts forward herein is to demonstrate the uniqueness of membrane-based microneedles to accomplish real-time, on-body monitoring of multiple ions simultaneously. The use of multi-ion detection is clinically relevant since it is expected to provide a more complete and reliable assessment of the clinical status of a subject concerning electrolyte disorders and others. We present a microneedle system for transdermal multiplexed tracing of pH, Na+, K+, Ca2+, Li+, and Cl–. The device consists of an array of seven solid microneedles externally modified to provide six indicator electrodes, each selective for a different ion, and a common reference electrode, all integrated into a wearable patch read in a potentiometric mode. We show in vitro measurements at the expected clinical levels, resulting in a fast response time, excellent reversibility and repeatability, and adequate selectivity. Close-to-Nernstian sensitivity, sufficient stability and resiliency to skin penetration guarantee the sensor’s success in transdermal measurements, which we demonstrate through ex vivo (with pieces of rat skin) and in vivo (on-body measurements in rats) tests. Accuracy is evaluated by comparison with gold standard techniques to characterize collected dermal fluid, blood, and serum. In the past, interstitial fluid (ISF) analysis has been challenging due to difficult sample collection and analysis. For ions, this has resulted in extrapolations from blood concentrations (invasive tests) rather than pure measurements in ISF. The developed microneedle patch is a relevant analytical tool to address this information gap.

Silicon rubber (Ecoflex 00-50 platinum cure, USA) and stainless-steel microneedles (MN) (Dermaroller local supplier, Sweden) were employed for the fabrication of the MN patch.
For the ex vivo experiments, rat skins of euthanized specimens at KERIC were cut into squared pieces and stored in the freezer at -18 °C. Before being used, each piece of skin was defrosted at room temperature and gently cleaned with distilled water. Micro-pH meter (LL, biotrode, Metrohm, Nordic Sweden), and ultra-micro pH meter (Orion, Ultra-Micro Combination pH Electrode, Thermo Scientific) were used for subcutaneous and extracted ISF pH measurements, respectively.

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ABL90 FLEX PLUS blood gas analyzer (Radiometer, Denmark) was employed for ions quantification in blood.

Fabrication of the MIMN patch.
The MIMN patch consisted of six MN-WEs (one for each analyte) and one MN-RE inserted on a silicon rubber subtrate. The MNs used in these experiments have a full length of 1.5 mm and a diameter of 250 μm. The patch substrate was fabricated by mixing equal volumes of the solutions, labelled as "Part A" and "Part B", in the commercial pourable silicon rubber kit (Smooth-on, USA) and filling a 3D printed mold of 23 mm of diameter and 1 mm of depth with the resulting mixture. Then, the substrate was allowed to cure for 3 h, according to the manufacturer instructions. Prior to the insertion into the substrate, the stainless-steel MNs were coated with carbon or Ag/AgCl ink for the working electrodes and the reference electrode, respectively. Afterwards, the MNs were attached on the substrate with Loctite Super Glue (Henkel Norden AB), which was left to dry at room temperature for 4 h. Finally, the functionalization with the ISMs was performed for each MN.
Working electrodes: Regardless of the analyte, the stainless-steel solid MN was coated with the carbon ink and the created film was allowed to cure in the oven at 120 °C for 10 min. Once room temperature is reached, the C-MNs were fixed in the substrate and the f-MWCNTs film was added by drop-casting (10 layers of 2 µL) a dispersion of f-MWCNTs in THF (1 mg/mL), with 4-min drying at room temperature in between layers. Finally, the corresponding ISM (composition collected in Table S1) was implemented by drop casting (3 layers of 1 µL), allowing each layer to dry 20 min at room temperature before the addition of the following one. The final film was dried for 4 h before conditioning. The working electrodes were conditioned overnight in 10 -2 M solution of the corresponding analyte. For the pH working electrode, it was conditioned in 10 -3 M of HCl. Analytical parameters were calculated according to IUPAC recommendations for ISEs. 4,5 Reference electrode: An Ag/AgCl film was deposited on top of the stainless-steel MN by dip coating of the commercial Ag/AgCl ink and, successively, the film was cured in the oven at 120 °C for 10 min. After appropriate fixation in the substrate, the reference membrane cocktail was drop-casted (3 layers of 3 μL) on top of the Ag/AgCl film. Each layer was allowed to dry at room temperature for 20 min before drop casting the next one. Then, the last layer was dried for 4 h before an overnight conditioning in 3 M KCl. Finally, the MN-RE was dried at room temperature for 1 h and a volume of 2 μL of polyurethane was drop casted on top of the MN and left it to dry for 4 h. The aim of this last layer was avoiding the salt (KCl) leaching out and improving the potential stability of the MN-RE. 6 Potentiometric measurements. Calibration experiments were carried out at room temperature (22 ± 1 °C) under constant stirring of 300 rpm (stirrer IKA COLOR SQUID S000, IKA, Germany). The MNs in the MIMN patch were connected to the potentiometer by a cable based on electrical clamps and BNC outputs. The activity coefficients to be used in the calibration graph were calculated using a two-parameter Debye Hückel approximation from the experimental concentrations. 7 Each logarithmic activity was plotted against the corresponding steady-state potential, and the curves were fitted to the Nernst equation.
Selectivity studies. Selectivity was evaluated using the separate solution method (SSM) according to Bakker et al. Individual calibration graphs were accomplished for the primary and the interfering cations, and the logarithmic selectivity were calculated by extrapolating the response to ! = " = 1 using the portion of the calibration curve close to Nernstian response.

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The use of the MIMN patch in euthanized rats. The potentials of the MNs in the MIMN patch were recorded with a hand-made potentiometric electronic board equipped with a Bluetooth low energy system able to take up to 900 samples per second.3 After the measurements, cardiac puncture blood collection was performed following established methods. After that, once rat backs were opened with a scalpel, pH was measured from the ISF in the exposed subcutaneous tissue by a micro-pH meter for pH measurements validation purposes. ISF and blood samples were additionally collected. Serum was obtained by placing the corresponding blood extraction tube in an upright position at room temperature to allow the blood to clot (15 -30 min). After that, the clot was removed by centrifuging at 1100 rpm for 10 minutes. For IC measurements, deproteinization was performed by mixing the serum with ethanol (31.5% v/v) for 30 minutes. The supernatant was removed after centrifugation at 1100 rpm for 5 minutes.
ISF collection in pieces of rat skin and euthanized rats. ISF was collected by using a homemade device consisting in a plastic hub containing 4 hollow microneedles (0.24 x 0.11 x 10 mm, Micropoint Technologies Pte Ltd, Singapore). The hollow length exposed in the hub was 1.5 mm and it was coupled with a microfluidic PFTE tubing (Sigma-Aldrich) with 0.3 and 0.6 mm of inner and outer diameter, respectively. A time of 10 min extraction was assisted by means of a tubing (Tygon LMT-55, ISMATEC, Cole-Parmer GmbH, Germany) connected to a peristaltic pump (ISMATEC IPC series, Cole-Parmer GmbH, Germany). ISF extraction was performed just after the sensing procedure with the MIMN patch to minimize the possible alterations of the ISF either in quantity or quality.  Tables   Table S1. Compositions of the ion-selective membranes (ISMs) cocktails. Values are expressed in mg of the compound solved in 1 mL of THF. Values in brackets show the concentration of the corresponding compound in mmol kg -1 of membrane.
a These ions did not display a Nerstian slope and therefore, the calculated logarithmic selectivity coefficients are "biased" and can be only interpreted qualitatively as "apparent" values. b Therapeutic values were used as the expected concentration. 25 c Values calculated from the higher concentration of the interfering ion that can be found in ISF.    1.0 (0.9) 0.80 ± 0.02 0.9 11 #14

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1.0 (0.9) 0.40 ± 0.03 0.5 20 #15 10.0 (9.5) 3.1 ± 0.7 3.5 11 #16 10.0 (9.5) 9.9 ± 0.9 11.1 11 Cl -#17 10.0 (8.9) 11.1 10.7 4 a Micro pH electrode for pH and IC for the rest of ions. b pH units. c % of difference between the result observed with the MIMN and that with the IC. a The pH could not be measured because the subcutaneous part of the back was dry when the measurement was attempted. Figure S1. SEM images of a cross-section of the WE for pH (left). Magnification of the cross-section (right).